Guanine-rich oligonucleotides can spontaneously self-assemble into four-stranded helices in vitro (Sen, D. & Gilbert, W., Nature 334:364-366 (1988); Kang, C. et al., Nature 356:126-131 (1992)). These four-stranded complexes can further associate into superstructures composed of 8, 12, or 16 oligomers (Sen, D. & Gilbert, W., Biochemistry 31:65-70 (1992)). In addition, some guanine-rich oligonucleotides can also assemble in an offset, parallel alignment, forming long "G-wires" (Marsh, T. C. & Henderson, E., Biochemistry 33:10718-10724 (1994); Marsh, T. C. et al., Nucleic Acids Research 23:696-700 (1995)). These higher order structures are stabilized by G-quartets that consist of four guanosine residues arranged in a plane and held together through Hoogsteen base pairings (FIG. 1A-2). At least three contiguous guanines within the oligomer are critical for the formation of these higher order structures (Sen, D. & Gilbert, W., Biochemistry 31:65-70 (1992)). Such guanine-rich sequences exist in HIV-1 RNA sequences (Awang, G. & Sen, D., Biochemistry 32:11453-11457 (1993)), immunoglobulin switch regions, and eukaryotic telomeres (Sen, D. & Gilbert, W., Nature 334:364-366 (1988)). It has been suggested that four-stranded DNAs have a variety of biological roles, such as inhibition of HIV-1 integrase (Mazumder, A. et al., Biochemistry 35:13762-13771 (1996)), formation of synapsis during meiosis (Sen, D. & Gilbert, W., Nature 334:364-366 (1988)), and telomere maintenance (Williamson, J. R. et al., Cell 59:871-880 (1989); Baran, N. et al., Nucleic Acids Research 25:297-303 (1997)).
Telomeres are implicated in pairing sister chromatids (Kolberg, R., J. NIH Research 9:24-26 (1997)), chromosome segregation in mitosis (Kirk, K. E. et al., Science 275:1478-1481 (1997)), formation of synapsis in meiosis, cell aging (Lundblad, V. & Szostak, J. W., Cell 57:633-643 (1989)), and tumorigenesis (Murnane, J. P. et al., EMBO J. 13:4953-4962 (1994)). Telomerase is thought to be essential for the maintenance of chromosome ends and, in particular, for the synthesis of telomeric DNA. However, certain telomerase knockout yeast strains can survive and generate a TG1-3 tail in a cell cycle-regulated manner (Lundblad, V. & Blackburn, E. H., Cell 73:347-360 (1993); Wellinger, R. J. et al., Cell 85:423-433 (1996)). Additionally, some human tumor cell lines without detectable telomerase activity maintain telomeric DNA (Bryan, T. M. et al., EMBO J. 14:4240-4248 (1995)). Studies of telomerase RNA knockout mice demonstrate that oncogenically transformed telomerase null mouse cells containing detectably shortened telomeres form tumors in nude mice (Blasco, M. A. et al., Cell 91:25-34 (1997)). All of the above indicate that additional mechanisms exist to maintain chromosome ends in vivo.
This invention is based on data that indicates that DNA polymerases appear to be able to recognize and template DNA synthesis from the non-Watson-Crick DNA structures that are proposed to exist at the telomere. This activity explains how immortalized cell lines lacking detectable telomerase activity and telomerase knockout organisms survive and maintain telomere length. DNA synthesis primed from the alternative DNA structures at the telomere provides an additional mechanism for maintaining the integrity of chromosome ends in vivo. It must be noted that short oligonucleotide primers were rapidly extended by the polymerase into several hundred base molecules. This mechanism probably involves alternative DNA base pairings to stabilize replication templates active in promoting expansion of a genome comprised of linear chromosomes and it may have provided a mechanistic scaffold for primitive genome expansion. This mechanism may also influence expansions and contractions occurring during replication of guanine-rich repeats (including triplets) within the genome.